The possibility of binding the thus formed TY-Ag system to Congo red (CR) and albumin (BSA) was determined. TY-Ag binding to CR provides for better nanosystem solubility and enables its targeted intracellular transport and binding to immune complexes. The binding of TY-Ag or CR-TY-Ag to albumin also protects the system against the uncontrolled release of silver ions. It will also allow the delivery of silver in a targeted manner directly to the desired site in the case of intravenous administration of such a system. In this study, the MIC (Minimum Inhibitory Concentration) and MBC  values of the TY-Ag or BSA-TY-Ag systems were determined in two reference strains (Escherichia coli and Staphylococcus aureus). The paper presents nanosystems with a size of about 40–50 nm, with an intense antibacterial effect obtained at concentrations of 0.019 mM. We have also discovered that TY-Ag free or complexed with BSA  inhibited cancer cells proliferation. TY-Ag complex diminished migration and effectively inhibited the T24 cell viability and induced apoptosis. On the basis of the obtained results, it has been shown that the presented systems may have anti-inflammatory and antitumor properties at the same time. TY-Ag or BSA-TY-Ag are new potential drugs and may become in future important therapeutic compounds in human urinary bladder carcinoma treatment and/or potent antimicrobial factors as an alternative to antibiotics.According to the World Health Organization report, the increasing antibiotic resistance of microorganisms is one of the biggest global health problems. The percentage of bacterial strains showing multidrug resistance (MDR) to commonly used antibiotics is growing rapidly. Therefore, the search for alternative solutions to antibiotic therapy has become critical to combat this phenomenon. It is especially important as frequent and recurring infections can cause cancer. One example of this phenomenon is urinary tract infections that can contribute to the development of human urinary bladder carcinoma. This tumor is one of the most common malignant neoplasms in humans. It occurs almost three times more often in men than in women, and in terms of the number of cases, it is the fifth malignant neoplasm after prostate, lung, colon, and stomach cancer. The risk of developing the disease increases with age. Despite the improvement of its treatment methods, the current outcome in the advanced stages of this tumor is not satisfactory. Hence, there is an urgent need to introduce innovative solutions that will prove effective even in the advanced stage of the disease. In our study, a nanosystem based on ionic silver (Ag According to the World Health Organization report, the increasing antibiotic resistance of microorganisms is one of the biggest global health problems ,2. It isEnterobacteriacae species (such as Klebsiella pneumoniae and Escherichia coli (E. coli) recorded in southern and south-eastern Europe were resistant to third-generation cephalosporins, fluoroquinolones, and aminoglycosides [Staphylococcus aureus (MRSA) strains, once associated mainly with hospital-acquired MRSA (HA MRSA), are now also responsible for the majority of community-acquired staph infections (CA MRSA). Genetic studies have shown that the strains causing these infections often have the SCCmec type IV chromosome, which is not usually found in hospital MRSA strains. Thus, we are dealing with a new threat, and not with infectious hospital strains that got into the outside community [The percentage of bacterial strains showing multidrug resistance (MDR) to commonly used antibiotics is growing rapidly. Therefore, there is an urgent need to develop new drugs to combat them ,4,5. In ycosides . Methiciommunity . One of ommunity . www.cancer.net portal, it is almost three times more prevalent in men than in women [Bladder cancer is diagnosed in more than 430,000 patients worldwide every year, making it the ninth most common malignancy. Referring to the data for the United States from 2020 on the in women . Unfortuin women . Bladderin women . The disin women .A long-term indwelling catheter use is also a risk factor for bladder cancer. Conversely, treatments for an already diagnosed bladder cancer are often associated with catheterization. Administration of drugs into the bladder also increases the risk of infection. Various types of intravesical chemotherapy are used. For example, thanks to the HIVEC system used immediately after the transurethral surgery to remove (resect) the tumor, the progression and recurrence of the disease are significantly reduced by about 2.5 times. Treatment is based on heating and recirculation of the chemotherapeutic between the apparatus and the inside of the bladder through the inserted catheter. Such action results in better penetration of the drug into cells, which results in greater effectiveness of the therapy. However, patients with HIVEC therapy exhibit symptoms of inflammation . Finding−13) because anions with Kd < 10−13 do not remove silver from the complex [+ ions interact with the functional groups of the proteins, e.g., thiol groups of proteins present in the cell membrane, resulting in their deactivation [+ ions alter the 3D structure of proteins upon interaction, interfere with disulfide bonds, and block active binding sites leading to overall functional defects in the microorganism [Therefore, the search for innovative solutions alternative to the commonly used antibiotic therapy has become a necessary requirement to combat the problem of microbial multidrug resistance, recurrent urinary tract infections, and urinary bladder carcinoma . A promi complex . Thanks tivation ,17. Bothorganism .3), silver complexed with TY (TY-Ag), its complex with albumin (BSA-TY-Ag), and the complexes with Congo red (CR) . CR is a supramolecular drug carrier, which additionally ensures a better solubility of the TY-Ag complex. Additionally, CR reduces silver toxicity and binds to antigen-bound antibodies. Therefore, research is underway on its use as a potential carrier factor for drugs, e.g., doxorubicin [The present work compares the effect of free silver  and Staphylococcus aureus . Free Ag+ ions show high antibacterial activity against Gram-positive and Gram-negative bacteria, but also against viruses and fungi [Escherichia coli, although other bacteria were also cultured (Klebsiella pneumoniae or Pseudomonas aeruginosa) [S. aureus may be a pathogen of the urinary tract among patients in long-term care facilities, wearing indwelling catheters, with urinary tract obstruction and/or malignant neoplasms, with instrumentation [S. aureus bloodstream infection (SABSI) from an “ascending” urinary tract source (UTS) [Two reference strains were selected for the microbiological analyzes carried out in this study: nd fungi . The maiuginosa) . S. aureentation . In thesce (UTS) ,26 may hThe characterization of the presented systems is the subject of this study. The size and stability of the resulting complexs were characterized by electrophoresis, gel filtration, UV-VIS spectrometry and DLS methods. The real effect of the added CR and BSA on the size of the systems and their effect on bacteria and neoplastic cells was assessed.+ to TY at different molar ratios of TY-Ag (1:1.6 and 1:0.8) was compared in order to optimize the complex for further use in microbiological analyzes. It has been shown that a strong TY-silver complex is formed, which at a ratio of 1:0.8 has no excess silver, does not decay in electrophoresis, and does not travel to the cathode (-).The aim of the analysis was to show that ionic silver can bind to Titan yellow and then can form complexes with CR, which improves solubility (CR-TY-Ag), and with BSA, which provides a protective role. The binding efficiency of AgFirst, the complexes formed on the basis of the TY-Ag complex were analyzed in a molar ratio of 1:1.6 . BSA comThe TY-Ag complex (When comparing samples 8 (CR-TY), 9 (CR-TY-Ag), and 10 (BSA-CR-TY-Ag), migration to the anode is clearly slowed down as the complexes are increased by adding further factors. This proves the formation of these complexes A. Sample+, it binds to TY or TY-CR in albumin (+—migration towards the cathode is visible (sample 6)  A.As in the electrophoresis presented above, at the ratio TY-Ag = 1:0.8, slight traces of streaking excess silver were visible; therefore, the target ratio of TY-Ag = 1:0.5 was used in microbiological experiments. It ensures that the added silver is completely bound, and a strong complex is obtained that does not decompose under electrophoresis. Thereby it is certain that the administration of excess free silver in microbiological tests has been avoided.The comparative analyzes of the UV/VIS spectra show that TY binds to BSA, as evidenced by the hypochromic effect at 392 nm wavelength, with a slight shift towards longer wavelengths and maintaining the unchanged maximum at 330 nm. Additionally, there is a maximum at 280 nm related to albumin complexation. The spectrum of TY-Ag or BSA-TY-Ag differs markedly from the spectrum of free TY or BSA-TY complex .The analysis shows that both TY and TY-Ag are bound to albumin and descend quickly (approx. 1 mL each) the column filled with BioGel P-150. The free TY descends more slowly, becoming smoother as it passes through the gel, and most of it is delayed by about 2.5–3 mL. This demonstrates the complexation of TY and the TY-Ag system by albumin .The autocorrelation curves of TY-Ag relative to free TY were analyzed. A shift in the autocorrelation curve of the TY-Ag complex relative to the autocorrelation curve of free TY was observed, which proves the presence of large TY aggregates formed under the influence of bound silver.H1 = 4.4 nm and RH2 = 26 nm) and monodisperse plot for the TY-Ag complex with one peak (RH = 41 nm). During the % mass analysis, there is a clear shift of the maximum for the TY-Ag complex (RH = 19.5 nm) compared to the free TY (RH = 2.4 nm). This proves the presence of TY-Ag aggregates . It was concluded that no large aggregates were formed and that TY was likely to bind at the albumin-binding cleft, and there was no bridging of albumin molecules  relative to the maximum for free BSA and BSA-TY . An additional maximum also appears in the % mass plot for the samples of the BSA-TY-Ag complex: RH1 = 3.6 nm and RH2 = 22.2 nm relative to the maximum for free BSA and BSA-TY (in both cases for % mass RH = 3.9 nm). This proves the presence of larger BSA-TY-Ag aggregates or the possibility of Ag+-mediated bridging of two albumin molecules (BSA-TY-Ag-TY-BSA). As a result, nanosystems containing albumin and silver are created, which are still relatively small.The appearance of an additional maximum was registered on the % intensity plot for the samples of the BSA-TY-Ag complex  was determined for the molar ratio of TY-Ag (1:0.5), where we can be sure that the silver is completely bound (well diffusion in agar experiment). The presence of albumin in the complex doubles the MIC value 0.039 mM for Gram-positive S. aureus, which has a thick bacterial envelope, and there is no increase in the MIC value for Gram-negative E. coli .The complexes with the TY-Ag molar ratio of 1:0.5 were analyzed, which, according to the results of the electrophoresis, were characterized by the complete binding of AgNOE. coli and S. aureus, the influence of the presence of TY on the enlargement of growth inhibitory zones can be seen. Free AgNO3 produces smaller zones of inhibition than the complexed Ag+. An increase in these zones was also observed after the addition of CR and after the addition of BSA. However, the simultaneous use of BSA and CR (quadruple complex: BSA-CR-TY-Ag) resulted in smaller zones than using CR and BSA alone, but still increased relative to the free Ag+. The use of CR and/or BSA has been shown to increase the solubility of TY-Ag and to provide protection, thus improving the antibacterial effect of silver against both Gram-positive bacteria as exemplified by S. aureus and Gram-negative bacteria as exemplified by E. coli (Both in the case of  E. coli .+ was 0.057 mM [The effect of TY-Ag or BSA-TY-Ag complexes on T24 cell proliferation (MTS), migration (scratch test) as well as viability/survival, and apoptosis induction  was assessed. The preliminary results obtained by us earlier on human U937 lymphoid cell line in which late apoptosis (73.65% of cells) and necrosis (about 25% of cells) was observed after 72 h, after using TY-Ag 1:0.8 (where the concentration of TY was 0.072 mM and concentration of Ag0.057 mM  providedE. coli and S. aureus strains after the addition of TY-Ag in a molar ratio of 1:0.5) as the concentration of Ag+ = 0.019 mM (19 µM). The addition of BSA in both cases increased the MIC value to an average of 0.036 mM for both analyzed strains (clearly for S. aureus and less unambiguously for E. coli). Free TY gave no zones of growth inhibition. Hence, for the analysis of T24 bladder tumor cell proliferation, concentrations of TY = 40 µM and 20 µM were selected, with which complexes with Ag+ (TY-Ag = 1:0.5 molar ratio) and in some experiments additionally with albumin (BSA-TY = 1:10 molar ratio) were prepared. The complexes were added to the culture medium at the Ag+ concentrations of 10 μM, 15 μM, and 20 μM. The cell proliferation analysis was performed by the MTS assay at 24, 48, and 72 h after the addition of the agents. The exposure of T24 cells to TY-Ag resulted in significant dose-dependent suppression of proliferation, evaluated with MTS assay, compared to the control cultures , as shown in The MIC values were determined in the previously described experiment (for + concentration of 10 µM did not reduce cell proliferation in the presence of complexed TY (20 µM). BSA at these concentrations of TY and Ag+ had practically no effect on proliferation. The combination of Ag+ (10 µM) with TY at a higher concentration (40 µM) reduced the proliferation to 89.6% of the control after 72 h of culture.It was observed that an Ag+ at a concentration of 15 µM. The complex with a lower concentration of TY in TY(20 µM)-Ag(15 µM) reduces the proliferation of cells after 72 h of culture to the level of 86%, and the protective effect of BSA is visible, which maintains proliferation at the level of 98.6%. However, the higher concentration of TY in TY(40 µM)-Ag(15 µM) significantly reduces the proliferation of cells after 72 h of culture to the level of 65.6% of control. When cultures were observed after the cultivation time of 24 h and 48 h, the clear protective role of BSA was visible, which manifested by the increase of cell proliferation from 75.8% to 100% and from 67% to 81% of control after 24 and 48 h of cultivation, respectively. Only after 72 h of incubation of T24 cells in the presence of the TY(40 µM)-Ag(15 µM) complex, both with and without BSA, the same inhibitory effect on the proliferation rate was observed, equal to 65% of the control. It can be concluded that the concentration of Ag+ = 15 µM in complexes with TY is a limiting concentration, and by regulating the concentration of TY or BSA, one can obtain complexes reducing the proliferation to a greater or lesser degree, depending on the needs.Much greater differences are visible after using Ag+ (20 µM) complexes, the proliferation decreased to the level of approx. 45% of control. At this concentration of Ag+, a protective role of BSA was also observed for both the concentrations of TY used (20 µM and 40 µM) -Ag(20 µM), proliferation drops to 63%. It is slightly elevated after using BSA in this complex. In contrast, in culture with TY(40 µM)-Ag(20 µM), proliferation decreased to 68%. BSA in this complex increases proliferation to 74%. After 72 h of cultivation with all the analyzed Agd 40 µM) .The concentration of TY used in each sample was 60 µM , while the concentration of silver was variable. Concentrations of TY-complexed silver of 15 µM and 30 µM (TY(60 µM)-Ag(30 µM) = 1:0.5 molar ratio; TY(60 µM)-Ag(15 µM) = 1:0.25 molar ratio) caused Cas 3 activation, indicating apoptosis induction. The other concentrations of silver complexed with TY, namely 7.5 µM and 3.75 µM (TY(60 µM)-Ag(7.5 µM) = 1:0.125 molar ratio; TY(60 µM)-Ag(3.75 µM) = 1:0.625 molar ratio) had no effect of Cas 3 activation, just as free TY .E. coli and S. aureus strains) for silver in the TY-Ag complex (1:0.5), can induce complete apoptosis of T24 cells (Using the flow cytometry method (Annexin/propidium iodide), apoptosis was assessed in the tumor cells of the bladder cancer cell line T24 after the addition of the TY(20 µM)-Ag(15 µM) complex. A total of 48 h after the addition of the factor, TY-complexed silver, added at a concentration of 15µM, resulted in a nearly complete apoptosis of 96.6%. This result confirmed that this concentration, close to the MIC value = 0.019 mM -Ag(1 µM) complex, and the cell migration was evaluated with a wound-healing test/scratch assay. A significant reduction in cell motility was observed upon exposure to TY(20 µM)-Ag(1 µM) . OpticalThe arrest of cell division may be the result of disturbances in the course of the cell cycle and/or induction of apoptosis and necrosis in cells. Therefore, in the next stage of the study, the influence of the tested compounds on the course of the cell cycle in the cultures of T24 cells after 48 h of incubation was determined. Cytometric analysis showed that the tested compounds (except for taxol) caused a slight increase in the number of cells in the G1 phase while reducing the number of cells in the S and G2 phases of the cell cycle .The prevention of infections is mainly associated with the use of antibiotics, the effectiveness of which is decreasing. In addition, frequent and recurring infections can cause cancer. For these reasons, finding an alternative, innovative therapy integrating antibacterial and anti-cancer treatment is the goal of scientific research ,27,28.Finding an agent that would combine the ability to minimize infections and anti-cancer activity seems to be very important. Such an agent could have practical applications in hospital treatments, and such attempts have long been made, but most involve alternative use of antibiotics. Various antibiotics are used in the treatment of neoplasms due to their anti-proliferative, pro-apoptotic, and anti-epithelial-mesenchymal transition (EMT) abilities . Yadav eE. coli and S. aureus. It is used to produce disinfectants, in dental composites, or as bactericidal coatings [Among non-antibiotic anti-infectious agents, silver has been known for a long time for its antibacterial and anti-inflammatory properties. Applied in the form of nanosilver, it is used in many areas of medicine. Plastic catheters coated with silver nanoparticles prevent biofilm formation by bacteria, including coatings . Silver coatings . The antcoatings ,37. Silvcoatings ,39. Due coatings . The maicoatings ,42, especoatings . AgNPs acoatings , as wellcoatings .AgNPs exhibit anti-cancer properties against various cancer cell lines, such as MCF-7 breast cancer cells , HCT116 + becomes better available both for bacteria to fight them and for bladder cancer cells. Our research, based on electrophoretic methods, gel chromatography, and UV/VIS spectrophotometry, also showed that the TY-Ag system could additionally be bound by albumin or by Congo red. As a result, complexes were obtained that not only bound ionic silver but also provided protection and better solubility. The TY-Ag complexes and the triple complexes of BSA-TY-Ag or CR-TY-Ag represent systems of potential therapeutic value. Due to the very good solubility provided by CR, as well as the protective effect of albumin, these systems can reduce the accumulation of metals in the body by facilitating the removal of silver from the complex. There are examples in the literature of the interaction of silver nanoparticles with proteins. Although the capping proteins can increase the internalization of AgNPs into cells via endocytosis, regulate cellular uptake and bioavailability of AgNPs, they can also be responsible for the toxic activity of AgNPs [Titan yellow comprises symmetric polar groups and aromatic rings. Its halves are linked by a tri-azo bond capable of complexing metal ions, particularly silver and mercury. The silver-containing complex is more convenient due to its stability and formation in both neutral and slightly alkaline environments ,55,56. Tof AgNPs . The intof AgNPs ,59. The Therefore, further studies are needed to understand the type of proteins and their precise role in the formation and bioactivity of nanoparticles.E. coli and S. aureus have been presented here for the first time. E. coli was selected because it is the main bacteria isolated from cases of urinary tract infections [S. aureus, a facultative anaerobic Gram-positive coccus, was selected for use in this study because it is one of the facultative bacteria found in inflammation of the urinary bladder and it can develop resistance to antimicrobial agents. S. aureus may be a pathogen of the urinary tract among patients staying in long-term care facilities, wearing indwelling catheters, with urinary tract obstruction and/or malignant neoplasms [Microbiological analyzes showing the effect of silver in various forms have been previously carried out on different bacterial strains . Neverthfections , whereaseoplasms .E. coli or S. aureus bacteria, while zones of growth inhibition are visible with the Ag+ complex, TY can be considered a useful type of carrier for silver. The results obtained by us have shown that thanks to the complexation of Ag+ with TY, a lower concentration of the administered silver can be used, which significantly reduces its toxicity and, additionally, provides protection. Additional protection is provided by albumin, which in the BSA-TY-Ag complex has increased the zones of inhibition of bacterial growth in both E. coli and S. aureus, as confirmed by our research. Additionally, the complexes with Congo red, which increases the solubility of the TY-Ag complex, increased the zones of inhibition of bacterial growth. The use of CR and/or BSA has been shown to increase the solubility of TY-Ag and to provide protection, thereby improving the antibacterial activity of silver in both Gram-positive bacteria, for example S. aureus, and Gram-negative bacteria, e.g., E. coli.Since our research has shown that free TY does not inhibit the growth of The relationship between the size of nanosystems and their antimicrobial efficacy (prokaryotic cells) is often analyzed in the literature. Similar to prokaryotic cells, in eukaryotic cells (antitumor efficacy), the size of AgNPs is a key factor determining their penetration into the cells by diffusion (translocation), endocytosis, or phagocytosis ,62,63. IS. aureus. [Different silver-based nanosystems may give different MIC and MBC results for the tested bacterial strains. This variability may be due to the methodology used to prepare the silver nanoparticles and the size of the silver nanoparticles used. A study by Ayala-Núñez NV et al. estimated MIC, MBC of 10 nm silver nanoparticles at a concentration of 1.35 mg/mL against  aureus. . The aut aureus. .E. coli and for S. aureus), and for BSA-TY-Ag at 0.019 mM for E. coli and 0.039 mM for S. aureus. MBC for TY-Ag (1:0.5) was found to be 0.036 mM (for E. coli) while the determination of MBC for S. aureus was impossible. MBC for BSA-TY-Ag was calculated as 0.07 mM(for E. coli), whereas the determination of MBC for S. aureus was impossible. It was reported that the activity of silver in AgNPs against Gram-negative bacteria was stronger than against Gram-positive bacteria [S. aureus, also increases MBC for E.coli . BSA also provides protection in bladder cancer T24 cell culture (reduces the cytotoxic effect of the TY-Ag system). It can be treated as an advantage if we consider the system with albumin as a drug carrier that will prolong the action of silver, causing its gradual release. This protein may also play a protective role, e.g., when administered intravenously.Based on these results, we also conducted a MIC and MBC analysis of our test systems to determine the antimicrobial efficacy of our 50 nm nanosystems. MIC values for TY-Ag (1:0.5) were estimated at 0.019 mM  than Gram-negative bacteria E. coli , which is different from the results obtained by us [Comparing our results with the results of some research teams, we noticed low values of the above parameters (at the level of a few µM), much lower than those obtained with the use of biologically or chemically synthesized silver nanoparticles . In our ed by us . TherefoDespite the strong antimicrobial activity of AgNPs and a wide range of biomedical applications, their use as therapeutic agents is limited due to their cytotoxicity to mammalian cells .+ concentration up to 15 μM. At the same time, among the tested bacteria, MIC values for TY-Ag were lower for Gram-negative bacteria, such as E. coli, as well as for Gram-positive bacteria, such as S. aureus (MIC = 19 μM). The use of the BSA-TY-Ag complex resulted in no changes in MIC values for Gram-negative bacteria, such as E. coli (MIC = 3 μg/mL), and resulted in higher MIC value for Gram-positive bacteria, such as S. aureus (MIC = 6 μg/mL). This result in the case of Gram-positive bacteria is more favorable than that obtained in the studies of Wypij M. et al. They investigated the cytotoxic effect of the silver biogenic AgNPs nanoparticles biosynthesized by strain SF23 on MCF-7 breast cancer cells and RAW 264.7 macrophages. The cell viability of both tested cell lines was significantly inhibited in the presence of AgNPs at a concentration of 16 μg/mL or higher, which gives similar results as those obtained for silver in our complex presented in this study on the cells of bladder cancer T24, with a significant degree of malignancy. AgNP concentration in the range of 16–64 μg/mL decreased the viability of RAW 264.7 cells from 42.2 to 14.2%, while in the case of MCF-7 cells, the viability of the measured cells decreased from 38 to 15.8% [According to , if the to 15.8% . Based o+ complexed at 9.8 μg/mL. Late apoptosis (73.65% of cells) and necrosis (about 25% of cells) were demonstrated after 72 h, after using TY-Ag 1:0.8 . TY-Ag complexes were characterized and analyzed for their antibacterial and cytotoxic activities. They can interact with protein, in particular albumin, which increases the biocompatibility and protects the system, or with Congo red, a supramolecular carrier that can provide for targeted transport. The TY-Ag complex is a promising compound for the treatment of bladder cancer as well as the treatment of bactericidal infections and could open up new avenues for new non-antibiotic treatment strategies for infections.To summarize, we have demonstrated for the first time that TY-Ag complex regulates proliferation, survival, and migration of T24 urinary bladder cells as well as inhibits bacterial growth of two reference strains, Titan yellow , Congo Red , silver nitrate , bovine serum albumin  were used.S. aureus  and among Gram-negative strains: E. coli . American Type Culture Collection  delivered these two prokaryotic bacteria strains.Reference strains recommended by EUCAST  were chosen among Gram-positive strains: Bladder cancer cell line  was purchased from ATCC . MTS proliferation test , assay for activity of Caspase-3 , Annexin V with propidium iodide for Flow Cytometry analysis  and PI/RNase Staining Buffer assay  were used to analyze the influence of the tested factors on the cells.3/0.9% KNO3 buffer, pH 8.2 to the appropriate concentration. It was boiled for 2 min at 100 °C to dissolve, then cooled. Initially, the molar ratio CR:TY = 1:2 (and 1:5—unpublished results) was calculated.TY and CR were dissolved in 0.05 M TRIS/HNO3 was prepared fresh in a dark tube. It was dissolved in 0.05 M Tris/HNO3/0.9% KNO3 buffer, pH 8.2. Initially, the molar ratios of TY-Ag = 1:1.6 (samples with excess silver) or 1:0.8  or 1:0.5  were calculated. To prepare the TY-Ag complex, AgNO3 solution was added to TY and incubated for 3 min.AgNOTo prepare complexes with albumin, a 10× excess of TY over BSA or a 10× excess of CR over BSA was initially calculated. Then, after addition of BSA and the buffer, the mixture was incubated for 10 min. Co-micelle CR-TY (both components with the same molar mass) was used at 12-fold excess over BSA.ElectrophoresisElectrophoresis was performed in 1% agarose gel . Proteins were fixed in the gel with picrate and then stained with bromophenol blue solution. CR was removed from the gel by reduction with sodium dithionite. The sodium dithionite also revealed the location of the silver after the separation. The differences in the speed of migration of individual complexes and the possibilities of silver binding depending on the molar ratio of TY-Ag (1:1.6 vs. 1:0.8) were assessed.Assessment of TY and TY-Ag binding by BSA—Spectrophotometry3 and spectroscopically using UV/VIS spectroscopy  in a wavelength range from 200 to 800 nm, at a resolution of 1 nm. The TY without silver nitrate was used as the control sample. The spectra of TY, TY-Ag, as well as BSA-TY and BSA-TY-Ag, were compared, and on that basis, the formation of complexes was assessed.The synthesis of the TY-Ag complex was observed visually by the color change of the TY from yellow to orange after adding AgNOAssessment of TY and TY-Ag binding by BSA - Gel filtration ChromatographyUsing the gel-filtration method, the speed at which the systems differing in molecular weights (TY and the BSA-TY complex in a molar ratio of 1:10) passed through the column filled with BioGel P-150 was compared. The separation was done in 0.01 M PBS pH 7.4 buffer. An 80µL sample was applied to the column filled with 4 mL of the BioGel P-150, then the separation was carried out. Spectrophotometric analysis of the migration rates of free TY, TY-Ag, and complexed with protein was performed. An observed low value of the elution volume indicates the formation of large and stable complexes. DLSMeasurement and comparison of the hydrodynamic radii of TY and TY-Ag, BSA and BSA-TY or BSA-TY and BSA-TY-Ag were carried out with a Dyna Pro dynamic light scattering (DLS) detector  with an incident laser beam at λ = 824 nm and a fixed scattering angle of 90°. Measurement of each sample was performed at 25 °C after 3 min incubation in the DLS instrument. Each sample was measured 4 times with 20 acquisitions lasting 10 sec each. Outliers were rejected from the analysis, and the results were averaged.MicroorganismsS. aureus and E. coli, reference strains recommended by EUCAST  were chosen as control strains for susceptibility testing and as control strains in quality control. These strains are S. aureus (ATCC® 29213™) and E. coli (ATCC® 35218™)—a strain producing TEM-1β-lactamase, which causes resistance to all β-lactam antibiotics.In order to evaluate the effect of silver complexed with TY or with the CR-TY or with BSA-TY systems, screening tests were performed on two model reference strains: Gram-positive and Gram-negative bacteria. The complexes were tested using strains from the collection of the American Type Culture Collection (ATCC). In the case of MIC determination. Well Diffusion in agar. Assay and growth conditionsE. coli  and S. aureus  were determined with the method of well diffusion of compounds in agar. For this purpose, appropriate bacterial suspensions were prepared according to the following methodology presented by Gibała et al.: 0.5 optical density (OD) in McFarland scale was made to receive 1.5 × 108 CFU (colony forming units)/ mL solution, then the cell suspension was diluted to 106 CFU mL−1. The prepared suspension was used for further studies, including the determination of MIC. The modified diffusion-disk screening method described by Gibała et al. was used to study the antimicrobial efficacy of TY-Ag (molar ratio 1:0.5) (or its complexes with BSA) by evaluating the visible growth of microorganisms in the agar [The minimum dilutions of the analyzed compounds that inhibited the growth of the microorganisms were denoted as minimum inhibitory concentration (MIC). The values of MIC for different kinds of complexes, TY-Ag and BSA-TY-Ag; for two kinds of strains, the agar . Serial MBC determinationE. coli  and S. aureus  were determined according to the method described by Gibała et al. [The values of minimum bactericidal concentration (MBC) for different kinds of comlexes, TY-Ag and BSA-TY-Ag; for two kinds of strains, a et al. .Cell Culture2 content and 95% humidity, changing the medium every 24–48 h. Cells were grown until the culture area was completely covered. Cells were then passaged using 0.05% trypsin.T24 human bladder cancer cells were used in the study. These are transitional cell carcinoma (TCC) cells. Cells were grown in McCoy’s culture medium  containing penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum . Cultures were carried out at 37 °C with 5% COTreatments of the Cells+ compound: 10 μM, 15 μM, and 20 μM and 2 concentrations of Titan Yellow (TY): 20 μM and 40 μM were analyzed in the cell proliferation assays. Then the selected concentrations were used for the evaluation of cell survival and migration.The Titan yellow (TY) complexed with silver (TY-Ag) and TY-Ag in combination with albumin (BSA) were added at 24 h from seeding the cells on the plate. The combination of 3 concentrations of AgCell Proliferation Assays3 cells/well. After 24 h growth at 37 °C in a humidified atmosphere (Day 0), the culture medium with the analyzed complexes (TY-Ag and BSA-TY-Ag) was added to the cells. The concentrations of TY of 40 µM and 20 µM were used for the preparation of the complexes with Ag+ (in the molar ratio TY-Ag = 1:0.5) and additionally with albumin (in the molar ratio BSA-TY = 1:10). The incubation was continued for the next 24, 48, and 72 h at 37 °C in a humidified atmosphere. The MTS assay  was used to determine the influence of the Ag+ complexes (TY-Ag or BSA-TY-Ag) on the proliferation of T24 cells. The absorbance was measured at 570 nm using an Epoch Microplate Spectrophotometer . Four replicate wells were used for each experiment. The effect of TY-Ag or BSA-TY-Ag complexes was presented as the proliferation curves [T24 cells were seeded on 24-well plates at a density of 15.25 × 10n curves ,83. Cell Survival Analysis+: 3.75 µM, 7.5 µM, 15 µM, and 30 µM complexed with TY (60 µM) for 24 and 48 h, then the cells were lysed in lysis buffer 0.05 M Tris/HCl, pH 7.6, 0.1 M NaCl, 0.001 M EDTA, 1% Triton X-100, and assayed for activity of Caspase-3 (DEVD-like caspase activity). An identical experiment was performed in parallel to estimate the cell number in each well (MTS). The amount of aminofluorocoumarin (AFC) released during incubation with Ac-DEVD-AFC was measured using a spectrofluorometer. For clarity of representation, fluorescent signal reflecting Caspase-3 activity was normalized to cell number (DEVD-like caspase activity/MTS cell number) and expressed as a fold increase in relation to untreated/control sample. The results show a single experiment performed in triplicates. The obtained data were averaged, and the standard deviation is represented in graphs by bars [Caspase-3 Activity Assay: Apoptosis was analyzed by evaluation of the Caspase-3 activity. Briefly, cells were seeded and cultured on 24-well plates with TY-Ag in 4 concentrations of Ag by bars .5) were seeded and cultured overnight on a six-well plate with TY and TY-Ag for 24 h. Approximately 1 × 105 cells were stained with Annexin V for 15 min at RT in the dark. After the incubation, 5μL of propidium iodide and 400 μL of buffer solution were added prior to the cytometric analysis alone. Fluorescence intensities of treated samples and controls were analyzed by flow cytometry on a BD FACSCantoTM . Experiments were performed at least twice.Flow cytometry: Apoptosis was analyzed with PE Annexin V Apoptosis Detection Kit I , following the manufacturer’s instructions. Briefly, T24 cells . The monolayer was scratched with a pipette tip and washed with phosphate-buffered saline (PBS) to remove floating cells. The scrape was monitored and photographed after 24 h incubation. The pictures were analyzed using Image J software , as described previously .Cell Cycle AssayCell cycle analysis was performed with the PI/RNase Staining Buffer assay . In this method, PI enters the cell through damaged membranes and can integrate into double-stranded DNA producing a fluorescent signal when properly excited. PI can also integrate into double-stranded RNA, therefore cells are treated with RNAse (RNA digestion). The amount of PI attached to the cell correlates with the amount of DNA, and thus allowed for the quantification of cells at particular phases of the cell cycle. For this purpose, cells, after 48 h of incubation, were rinsed with PBS and then fixed with cold 70% ethanol for 2 h. The cells were then washed again with PBS and resuspended in staining buffer  for 15 min in the dark. Acquire data on a flow cytometer . The analysis of the cell cycle was carried out in the ModFit program.To conclude, our results suggest that TY-Ag and BSA-TY-Ag, also with CR, are promising complexes in antimicrobial therapy and in the treatment of human urinary bladder carcinoma during inflammation and cancer.The complexes can be effective in the removal of the bacterial film. TY-Ag or BSA-TY-Ag are new potential drugs and may become important therapeutic options in human urinary bladder carcinoma treatment or/and important antimicrobial agents as an alternative to antibiotics. Treatment of T24 cells with such inhibitors diminishes proliferation and migration of T24 cells and induces apoptosis. The ability to bind TY-Ag to BSA will protect the drug from uncontrolled release and ensure targeted delivery directly to the desired site when such a system is intravenously administered. Binding to CR will increase the solubility of the system as well as enable targeted transport using antigen-antibody systems. Based on our findings, it can be concluded that the TY-Ag complex may be a potential cytotoxic agent against cancer cells and bacteria. Considering the potential of these complexes, they may be recommended for biomedical applications after further in vivo/clinical studies.


N-acetyl-l-cysteine (NAC) and crosslinked via UV-assisted thiol-ene addition, in order to obtain efficient membranes able to resist bacterial adherence and biofilm formation. These membranes were subjected to in vitro testing for microbial adherence against S. pneumoniae using standardized tests. WISTAR rats were implanted for 4 weeks with crosslinked siloxane samples without and with NAC. A set of physical characterization methods was employed to assess the chemical structure and morphological aspects of the new synthetized materials before and after contact with the microbiological medium.Within this study, new materials were synthesized and characterized based on polysiloxane modified with different ratios of  Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa [Cochlear implants are implantable medical devices that provide the chance to (re)habilitate hearing in deaf children and adults who have no benefit from conventional hearing aids. The importance of hearing in language development, verbal communication and social integration imposes cochlear implantation for those who suffer from bilateral severe profound congenital hearing loss, especially at younger and younger ages . The curruginosa . All of ruginosa .Within this context, the most common strategies applied are the (1) antibiotic coating of implants and (2) covalent attachment of antimicrobial molecules on the surface of the implant to prevent bacterial adhesion, biofilm development, and avoidance of drug resistance to commonly used antibiotics .The latest strategy used active molecules that are immobilized by covalent bonds on the implant surface, obtaining a bioactive implant that induced a decrease of the local toxicity and high resistance to bacterial colonization and biofilm formation . This meSuch issues, presented above, also occur in the case of biointegrated electronics in vivo. Biointegrated electronics are widely applied for various in vivo applications, such as cochlear or cerebral implant , which sN-acetyl-l-cysteine (NAC) with antibiofilm activity were signaled in the literature [N-acetyl-l-cysteine has weak antibacterial activity, but it is a strong biofilm inhibitor [Different approaches have been proposed for a new generation of silicone materials that would give long-term antibacterial activity and prevent biofilm formation, preserving their biocompatibility. For example, recently, grafted hydrophilic polysiloxanes with terature ,17. It inhibitor . The mosnhibitor . To overN-acetyl-l-cysteine and different degrees of crosslinking. It is important to note that the crosslinker is a siloxane copolymer, synthesized by us, containing thiol (SH) functional groups grafted on the main siloxane chain. The NAC grafting and the crosslinking reactions are based on thiol-ene photoaddition, following the method described previously [S. pneumoniae, and the results were compared with a silicone material without NAC, synthesized in the same manner as those with cysteine. In vivo tests were done on WISTAR rats implanted for 4 weeks with crosslinked siloxane samples without and with NAC. The prepolymers were characterized by 1H-NMR and the crosslinked polysiloxanes were characterized by different techniques, such as Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), wettability, and Raman techniques.In the present work, we present the synthesis of a series of new siloxane materials based on crosslinked polysiloxane bearing different contents of eviously . The croThe synthesis of NAC-modified dimethyl-methylvinylsiloxane copolymers with different percentages of vinylmethysiloxane units  was performed by thiol-ene photoaddition initiated by DMPA, following the method described previously ,21, acco−1 (PDI = 1.5).As a model for structural characterization, all vinyl groups of ViMSi-10 with 10% of vinylmethysiloxane units  remained unchanged.In ectively . In addi1H-NMR spectrum of PNAC-10-100  containing different percentages in NAC and CoSH crosslinker copolymer were submitted to in vitro tests against an S. pneumoniae cells. It can also be observed that the viability of S. pneumoniae cells decreased in the series CR-PNAC-18-75 > PNAC-10-100 > CR-PNAC-18-30 > CR-PNA-18-0, indicating that even though the degree of crosslinking decreased with the increase of the content in NAC, the inhibition of S. pneumoniae cell proliferation is around 50% in the case of CR-PNAC-18-75 sample. It can also be concluded that with the increase of NAC percentage, the material became more efficient against S. pneumoniae cells. Taking into consideration these observations, the CR-PNAC-18-75 crosslinked polysiloxane sample was selected to be submitted to a more complex evaluation and to be compared with CR-PNA-18-0 as a control sample.From the MTS assay  performeS. pneumoniae cells that may appear due to the biological attachment of cells after 4 weeks of incubation.In terms of XRD analysis , all recMoreover, the investigation of different modifications in the thermal transitions of the discussed materials (CR-PNAC-18-0 and CR-PNAC-18-75) was evaluated with the DSC method. The recording of the heat flow as a function of temperature induced composition-dependent transitions in, but not limited to polymer-based structures, playing a very important role in elucidating different material aging behaviors  ,23,24,25S. pneumoniae.S. pneumoniae, ranging from around 0 °C to 200 °C, is due to moisture removal. From S. pneumoniae exhibited a glass transition temperature (Tg) domain centered at −122 °C, typical for polysiloxanes. Thus, the Tgs of these two samples reproduced almost perfectly, with no differences between them. Compared to the CR-PNAC-18-0 sample, the CR-PNAC-18-75 membrane before and after being incubated for 4 weeks with S. pneumoniae exhibited higher Tg values on the first heating curves, i.e., −115 °C. The fact that the Tg values registered for CR-PNAC-18-75 untreated and treated samples were identical on each heating curve confirms the homogeneity of the material. It should also be mentioned that single Tg values were registered, showing the absence of macroscopic phase separation, while the higher value as compared to pure PDMS-based materials is an additional confirmation of chemical modification with NAC. Both initial and treated CR-PNAC-18-0 samples exhibited an exothermic profile which reproduced at 211 °C and with similar enthalpy values (ΔHcr = −1.51 J g−1 and −1.451 J g−1), most probably correlated with the crosslinking of traces of residual vinyl groups. The weak and almost perfectly reproducible exothermic profile seems to not have affected the Tg on the second heating of both systems (−122 °C). A different situation was noted for the initial and treated CR-PNAC-18-75, showing complex exothermic profiles at high temperatures (155–222 °C), which might be correlated with both thermal crosslinking of residual vinyl groups, and supplementary physical crosslinking by H-bonding favored by the increased chain mobility. For the initial CR-PNAC-18-75, two distinct exothermic profiles appeared at 155 °C and 221 °C with the corresponding ΔHcr values of −2.324 J g−1 and −7.095 J g−1. For CR-PNAC-18-75 exposed to S. pneumoniae, the two exothermic peaks merged into a single profile with the first peak shifting significantly from 155 °C to 209 °C (ΔHcr = −12.11 J g−1). The additional crosslinking would explain the higher Tg values registered on the second heating curves of the CR-PNAC-18-75 samples (−109 °C). On the first DSC heating curves of CR-PNAC-18-75 before and after exposure to S. pneumoniae, endothermal peaks were registered above 250 °C . However, these peaks were affected by thermal degradation. Moreover, no component of the materials has such a high melting temperature, and the presence of non-bonded NAC is excluded based on the XRD spectrum showing no crystalline compounds. In addition, no corresponding crystallization profile was observed on the CR-PNAC-18-75 cooling curve. Rather, a possible explanation could be the breaking of the H-bonds. Furthermore, there was a significant difference, in both temperature and enthalpy, between the endothermal degradation profiles of initial CR-PNAC-18-75  and CR-PNAC-18-75 treated with S. pneumoniae . Such an important difference in thermal behavior between the sample before and after exposure to S. pneumoniae clearly indicates some kind of interaction of the copolymer with the culture medium triggered by the NAC organic groups. Therefore, it is most likely that the thermal degradation of the polysiloxane chains of CR-PNAC-18-75 was catalyzed by a component from the culture medium since different kinds of impurities are known to influence the polysiloxanes thermal degradation [The broad endothermic peak present on the first heating curves of samples CR-PNAC-18-75 and CR-PNAC-18-75 treated with radation .S. pneumoniae is proven by XRD analysis. Thus, we suppose that the “impurities” retained on the surface might generate, during heating, species which would have a catalytic role in enhancing the thermal degradation.Overall, the analysis of the thermal behavior of the synthesized samples has no significance for the envisaged practical application, but it can show indirectly that the biological testing medium affected the behavior of the material during heating. The fact that enzymes degrade NAC is well established , while ta = 95.1° and θr = 57.8° . Both the relatively low θa for a silicone-based material and larger hysteresis (CAH = 37.3°) were normal, given the chemical modification with a hydrophilic group. The surface free energy (γS) can be calculated from the contact angles [L is the surface tension of water, i.e., 72.8 mN·m−1.The different behavior of the modified films in presence of bacteria is surely influenced by the surface properties, i.e., contact angles and surface energy. Indeed, the dynamic water contact angle of the CR-PNAC-18-75 sample was measured by tensiometry (Wilhelmy method) and the found values were lower than those of pure PDMS, i.e., advancing and receding contact angles θt angles  with Equ−2), significantly increased compared with PDMS (16.5 mJ·m−2) [−1 [The obtained value for CR-PNAC-18-75 was 24.7 (mJ·m mJ·m−2) . The surm−2) [−1 . This co weighing about 280–300 g each . Under the same conditions, the CR-PNAC-18-0 control sample was also tested in vivo.Following the results obtained from the in vitro test, it was observed that the best efficacy against S. pneumoniae in vitro and in vivo after implantation for 4 weeks on WISTAR rats are presented in S. pneumoniae cells in vitro and in vivo for 4 weeks presented different amounts of bacteria on the surface. In this context, CR-PNAC-18-0 samples (SEM images obtained on the surfaces of the control sample (CR-PNAC-18-0) and CR-PNAC-18-75 sample before and after incubation with S. pneumoniae in vitro and in vivo , maybe as a result of the growth of the bacterial layer [Using the EDX analysis of CR-PNAC-18-0 and CR-PNAC-18-75 sample surfaces , it was al layer . After iIt is well known that, in silicone-base materials, the surface is generally enriched in Si. The very abrupt diminishment of the Si content of CR-PNAC-18-0 especially after in vivo test is in agreement with the SEM observation of biofilm covering most of the surface. In contrast, the NAC-modified polymer presented a less pronounced variation of Si content, again confirmed by SEM. Thus, the increase in S and O content in this sample after in vitro and in vivo experiments is mainly ascribed to changes in molecular conformation of the polymer at the surface during prolonged exposure to a wet environment.N-acetyl-l-cysteine , diethoxydimethylsilane ,dimethoxy-(3-mercaptopropyl)methylsilane , THF , and methanol  of high purity were used as received. Purolite CT-175 (sulfonated styrene-divinylbenzene cross-linked copolymer cationic exchange resins (St-DVB) with exchange capacity of 1.87 meq mL−1 was purchased from Viromet S.A. Dimethyl-methylvinylsiloxane copolymers (ViMSi) with 10 or 18 mol% methylvinylsiloxane units were prepared by a previously reported procedure [2,2-dimethoxy-2-phenylacetophenone , rocedure ,36,37.In a reaction vessel, 4.75 mL diethoxydimethylsilane, 0.25 mL dimethoxy-(3-mercaptopropyl)methylsilane, and 1 mL of distilled water were introduced and the mixture was purged with argon. Then, 0.1 g of St-DVB ion exchanger was added and the reaction was carried out at 50 °C for 4 h, then at room temperature for another 20 h. The cation exchanger was filtered out and the water and formed alcohols were removed by vacuum distillation. The resulted 1.5 g of CoSH copolymer a was kepFor obtaining crosslinked films, partial modification of ViMSi with different amounts of NAC was carried out in the first step b.The PNAC-X-Y copolymers  were obtained similarly, as described below.1H-NMR and FT-IR.A measure of 0.5 g (6.65 mmol) ViMSi copolymer with 10 mol% vinylmethylsiloxane units , δ (ppm): 0.15 (–SiCH3), 0.96 (-SiCH2-), 2.02 (-CH3), 2.73, 2.93 (-CH2SCH2-), 4.7 (-CH-).A measure of 0.52 g (6.8 mmol) ViMSi copolymer with 18 mol% vinylmethylsiloxane units . To obtain crosslinked films, the remaining vinyl groups of PNAC-18 with different amounts of NAC were crosslinked with CoSH.Preparation of crosslinked film: CR-PNAC-X-Y  crosslinked polymers were obtained by the same procedure as described above, using the appropriate amounts of NAC and CoSH.Streptococcus pneumoniae ATCC49619. The bacterial strain was refreshed in trypticase soy broth with defibrinated sheep blood for 24 h at 37 °C. The test surfaces were prepared as follows: each sample was placed in a Petri dish and sterilized at 254 nm for 15 min on each side.The antimicrobial efficacy of the materials was investigated by using in vitro adapted methods. A slightly modified Japanese industrial standard JIS Z2801:2000 which is based on a logarithmic number of live bacteria after 24 h of incubation was used, but in this case, the samples were incubated for 4 weeks, using a covering film to keep the thickness of the bacterial suspension. The antibacterial activity was determined against 2. After incubation, the samples were rinsed repeatedly and pipetted to 1.5 mL Eppendorf tubes. In order to quantify the bacterial anti-adherence properties CellTiter 96sAQueous One Solution Cell Proliferation Assay, MTS (Promega) was performed, by following the protocol recommended by the manufacturer, using the solutions resulting from the previous experiment incubated in 96-well culture plates and incubated for 24 h at 37 °C. MTS Assay Kit uses a colorimetric method for the sensitive quantification of viable cells. The MTS reagent was then added to each well and, after 4 h incubation, the absorbance at 490 nm was recorded with an EnVisions plate reader (PerkinElmer). The experiment included four replicates and was repeated three times. All data were expressed as the mean ± standard deviation (SD). Statistical analysis was performed XLSTAT software [The microbial suspensions were prepared with the reference strains in sterile solution to obtain turbidity optically comparable to that of 0.5 McFarland standards. Volumes of 0.2 mL from each inoculum were spread on the Petri dishes with trypticase soy broth with defibrinated sheep blood. The sterilized samples were placed on the plates, covered, and then incubated at 37 °C for 4 weeks in an atmosphere with 5% COThe experiments were conducted on young WISTAR female rats. Seven young female rats (mean age ≅ 4–5 weeks) weighing about 280–300 g each were implanted. Out of those seven experimental animals, five of them were implanted with CR-PNAC-18-0 and two of them with CR-PNAC-18-75. Every CR-PNAC fragment was presterilized using the ethylene oxide sterilization method. The animals were anesthetized with ketamine (4 mg/100 g intramuscularly) and xylazine (1 mg/100 g intramuscularly). The rat was positioned in left lateral decubitus and the retroauricular region was shaved and cleaned with 10% povidone. The dissection for the auditory bullae was performed by a retroauricular approach. It is established that the posterior term describes the caudal direction and the anterior term describes the rostral direction. The incision comprised the skin layer and measured approximately 5 cm. The auricle was slightly pinched and oriented anteriorly. The first anatomical elements used as landmarks were the trapezius muscle, the great auricular nerve, and the parotid gland. These elements were easily seen through the transparency of the muscle fascia. It was necessary to palpate the right mandibular angle, thus easily identifying the parotid gland that hides the path of the facial nerve. The trapezius muscle was pushed posteriorly to expose the main trunk of the facial nerve. Specific care was required manipulating the trapezius muscle due to the presence at this point of a large blood vessel. The trajectory of the facial nerve was followed in the caudal direction until the periosteum of the auditory bullae was observed anteroinferior of the nerve.Once the auditory bullae was accessible, the periosteum that covers the structure was stripped and then an opening was performed to gain access to the auditory bullae of the rat. To obtain access to the bullae, we used an otologic drill with a diamond head (1–3 mm in diameter). Subsequently, we prepared a socket for the test material that was comprised of musculocutaneous tissue placed on the back of the cervical area in connection with the created opening of the bullae. CR-PNAC-18-0 and CR-PNAC-18-75 were implanted in the pre-created musculocutaneous pocket, where the receptor-stimulator part of the device would normally be placed. After 4 weeks, all the samples were removed and submitted to morphological and physicochemical characterization.Surface morphology and elemental composition of the synthesized samples were evaluated with the help of a Verios G4 UC scanning electron microscope  (SEM), equipped with an energy dispersive X-ray spectroscopy analyzer  (EDX). After in vitro and in vivo incubation, the samples were inactivated at 121 °C for 15 min and then coated with 10 nm platinum using a Leica EM ACE200 Sputter coater to provide electrical conductivity and to prevent charge buildup during exposure to the electron beam. SEM investigations were performed in high vacuum mode using a secondary electron detector  at an accelerating voltage of 5 kV.X-ray diffraction analysis was performed on a Rigaku Miniflex 600 diffractometer using CuKα-emission in the angular range 3–90° (2θ) with a scanning step of 0.01° and a recording rate of 4°/min.−1 flow rate). The samples were heated and cooled at rates of 10 and −10 °C min−1.The DSC analyses were conducted on a DSC 200 F3 Maia device . Around 5 mg of each sample were weighed and placed into aluminum crucibles. The crucibles were sealed shut with pierced lids. The measurements were undertaken in a nitrogen atmosphere .Raman spectra of samples, before and after microbial exposure, were recorded using an InVia Raman microscope  in the 150–3200 cmDynamic Contact Angle Measurements were done by the Wilhelmy method using a Sigma KSV700 Automatic Tensiometer .3 at room temperature on a Bruker NEO-1 400 MHz spectrometer equipped with a 5 mm four nuclei (1H/13C//29Si) direct detection probe.NMR measurements were done in CDCl−1 by accumulation of 32 scans, at a resolution of 2 cm−1.FT-IR spectra were registered on a Bruker Vertex 70 spectrometer  in transmittance mode in the range 4000–400 cmStatistical analysis was done byusing XLSTAT | Statistical Software for Excel Available online .S. pneumoniae and in vivo tests on WISTAR rats for 4 weeks. It should be noted that as the structure of crosslinked polysiloxane contains a high NAC content, the material inhibits the formation of biofilms and decreases the number of bacteria adhering to its surface in both cases after in vitro exposure to S. pneumoniae or in vivo after implantation in rats for 4 weeks. Furthermore, it should be underlined that the structure of crosslinked polysiloxane without NAC is not affected after exposure to bacterial cells, while the materials with NAC in their composition are degraded maybe due to the enzymatic process of NAC. These features were demonstrated through XRD, DSC, and Raman methods.Crosslinked new polysiloxanes based-materials with different contents in NAC and different degrees of crosslinking were prepared by a green process, meaning thiol-ene photoaddition initiated by DMPA. The obtained materials were submitted for in vitro tests against After in vivo tests, when the CR-PNAC-18-75 sample was implanted on WISTAR rats for 4 weeks, the material surface showed bacteria in very small numbers and no biofilm formation was observed, making CR-PNAC-18-75 silicone material suitable for coating protection of biomedical devices, including cochlear devices.